Bleaching process comprising use of phenol oxidizing enzyme, a hydrogen peroxide source and an enhancing agent

ABSTRACT

The present invention relates to a process for providing a bleached look in the color density of the surface of dyed fabric, especially cellulosic fabric such as denim, comprising use of a phenol oxidizing enzyme such as a peroxidase or a laccase, a hydrogen peroxide source and an enhancing agent represented by formula (I). ##STR1##

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK95/00417 filed Oct. 18, 1995 and claims priority under 35 U.S.C.119 of Danish application 1217/94 filed Oct. 20, 1994, the contents ofwhich are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a process for providing a bleached lookin the colour density of the surface of dyed fabric, especiallycellulosic fabric such as denim.

BACKGROUND ART

The most usual method of providing a bleached stone-washed look in denimfabric or jeans is by washing the denim or jeans made from such fabricin the presence of pumice stones to provide the desired localizedlightening of the colour of the fabric. This is then followed by ableaching process where the fabric is treated with sodium hypochloriteat 60° C. and pH 11-12 for up to 20 min., followed by a neutralisationstep and a rinsing. Use of hypochlorite is undesirable, both becausechlorite itself is undesirable and because the neutralisationsubsequently generates high amounts of salts leading to disposal andpollution problems.

Bleaching enzymes such as peroxidases together with hydrogen peroxide oroxidases together with oxygen have also been suggested for bleaching ofdyed textiles (see WO 92/18683), either alone or together with a phenolsuch as p-hydroxycinnamic acid, 2,4-dichlorophenol, p-hydroxybenzenesulphonate, vanillin or p-hydroxybenzoic acid. The disclosed process isnot efficient as can be seen from Example 1 of the present invention.

Thus there is still a need for providing a bleached look in dyedfabrics. The problem to be solved is not easy as many VAT-dyes,especially indigo, are not soluble in water and have a very compactstructure on the fibre surface, making them difficult for an enzyme toattack.

SUMMARY OF THE INVENTION

Surprisingly it has been found that it is possible to create a veryefficient process for providing a bleached look in the colour density ofthe surface of dyed fabric, the process comprising contacting, in anaqueous medium, a dyed fabric with a phenol oxidizing enzyme system andan enhancing agent of the following formula: ##STR2## in which formula Ais a group such as --D, --CH═CH--D, --CH═CH--CH═CH--D, --CH=N--D,--N═N--D, or --N═CH--D, in which D is selected from the group consistingof --CO--E, --SO₂ --E, --N--XY, and --N⁺ --XYZ, in which E may be --H,--OH, --R, or --OR, and X and Y and Z may be identical or different andselected from --H and --R; R being a C₁ -C₁₆ alkyl, preferably a C₁ -C₈alkyl, which alkyl may be saturated or unsaturated, branched orunbranched and optionally substituted with a carboxy, sulfo or aminogroup; and B and C may be the same or different and selected from C_(m)H_(2m+1) ; 1≦m≦5.

DETAILED DESCRIPTION OF THE INVENTION Dyed Fabric

The process of the invention is most beneficially applied tocellulose-containing fabrics, such as cotton, iscose, rayon, ramie,linen, Tencel, or mixtures thereof, or mixtures of any of these fibres,or mixtures of any of these fibres together with synthetic fibres suchas mixtures of cotton and spandex (stretch-denim). In particular, thefabric is denim. The process of the invention may also be applied toother natural materials such as silk.

The fabric may be dyed with vat dyes such as indigo, or indigo-relateddyes such as thioindigo.

In a most preferred embodiment of the process of the invention, thefabric is indigo-dyed denim, including clothing items manufacturedtherefrom.

Phenol Oxidizing Enzyme Systems By the term "a phenol oxidizing enzymesystem" is meant a system in which an enzyme, by using hydrogen peroxideor molecular oxygen, is capable of oxidizing organic compoundscontaining phenolic groups. Examples of such enzymes are peroxidases andoxidases.

If the phenol oxidizing enzyme system requires a source of hydrogenperoxide, the source may be hydrogen peroxide or a hydrogen peroxideprecursor for in situ production of hydrogen peroxide, e.g. percarbonateor perborate, or a hydrogen peroxide generating enzyme system, e.g. anoxidase and a substrate for the oxidase, or an amino acid oxidase and asuitable amino acid, or a peroxycarboxylic acid or a salt thereof.Hydrogen peroxide may be added at the beginning of or during theprocess, e.g. in a concentration corresponding to 0.001-25 mM H₂ O₂.

If the phenol oxidizing enzyme system requires molecular oxygen,molecular oxygen from the atmosphere will usually be present insufficient quantity. The enzyme of the phenol oxidizing enzyme systemsmay be an enzyme exhibiting peroxidase activity or a laccase or alaccase related enzyme as described below.

According to the invention the concentration of the phenol oxidizingenzyme in the aqueous medium where the localized variation in the colourdensity of the surface of the dyed fabric is taking place, may be0.001-10000 μg of enzyme protein per g denim, preferably 0.1-1000 μg ofenzyme protein per g denim, more preferably 1-100 μg of enzyme proteinper g denim.

Peroxidases and Compounds possessing Peroxidase Activity

Compounds possessing peroxidase activity may be any peroxidase enzymecomprised by the enzyme classification (EC 1.11.1.7), or any fragmentderived therefrom, exhibiting peroxidase activity, or synthetic orsemisynthetic derivatives thereof (e.g. porphyrin ring systems ormicroperoxidases, cf. e.g. US 4,077,768, EP 537,381, WO 91/05858 and WO92/16634).

Preferably, the peroxidase employed in the method of the invention isproducible by plants (e.g. horseradish or soybean peroxidase) ormicroorganisms such as fungi or bacteria. Some preferred fungi includestrains belonging to the subdivision Deuteromycotina, classHyphomycetes, e.g. Fusarium, Humicola, Tricoderma, Myrothecium,Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia,Cladosporium or Dreschlera, in particular Fusarium oxysporun (DSM 2672),Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO 6113),Verticillum alboatrum, Verticillum dahlie, Arthromvces ramosus (FERMP-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli orDreschlera halodes.

Other preferred fungi include strains belonging to the subdivisionBasidiomycotina, class Basidiomycetes, e.g. Coprinus, Phanerochaete,Coriolus or Trametes, in particular Coprinus cinereus f. microsporus(IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g.NA-12) or Trametes (previously called Polyporus), e.g. T. versicolor(e.g. PR4 28-A).

Further preferred fungi include strains belonging to the subdivisionZygomycotina, class Mycoraceae, e.g. Rhizopus or Mucor, in particularMucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales,e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoyiolaceus(IFO 12382) or Streptoverticillum verticillium ssp. verticillium.

Other preferred bacteria include Bacillus pumilus (ATCC 12905), Bacillusstearothermophilus, Rhodobacter sphaeroides, Rhodomonas palustri,Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonasfluorescens (NRRL B-11).

Further preferred bacteria include strains belonging to Myxococcus, e.g.M. virescens.

The peroxidase may furthermore be one which is producible by a methodcomprising cultivating a host cell transformed with a recombinant DNAvector which carries a DNA sequence encoding said peroxidase as well asDNA sequences encoding functions permitting the expression of the DNAsequence encoding the peroxidase, in a culture medium under conditionspermitting the expression of the peroxidase and recovering theperoxidase from the culture.

Particularly, a recombinantly produced peroxidase is a peroxidasederived from a Coprinus sp., in particular C. macrorhizus or C. cinereusaccording to WO 92/16634, or a variant thereof, e.g., a variant asdescribed in WO 94/12621.

In the context of this invention, peroxidase acting compounds compriseperoxidase active fragments derived from cytochromes, haemoglobin orperoxidase enzymes, and synthetic or semisynthetic derivatives thereof,e.g. iron porphins, iron porphyrins, and iron phthalocyanine andderivatives thereof.

Determination of Peroxidase activity: 1 peroxidase unit (PODU) is theamount of enzyme that catalyzes the conversion of 1 μmol hydrogenperoxide per minute at the following analytical conditions: 0.88 mMhydrogen peroxide, 1.67 mM2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate), 0.1M phosphatebuffer, pH 7.0, incubated at 30° C., photometrically followed at 418 nm.

Laccase and Laccase Related Enzymes

In the context of this invention, laccases and laccase related enzymescontemplate any laccase enzyme comprised by the enzyme classification(EC 1.10.3.2), any chatechol oxidase enzyme comprised by the enzymeclassification (EC 1.10.3.1), any bilirubin oxidase enzyme comprised bythe enzyme classification (EC 1.3.3.5) or any monophenol monooxygenaseenzyme comprised by the enzyme classification (EC 1.14.99.1).

The laccase enzymes are known from microbial and plant origin. Themicrobial laccase enzyme may be derived from bacteria or fungi(including filamentous fungi and yeasts) and suitable examples include alaccase derivable from a strain of Aspergillus, Neurosnora, e.g. N.crassa. Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus,Trametes (previously called Polyporus), e.g. T. villosa and T.versicolor, Rhizoctonia, e.g. R. solani, Coprinus, e.g. C. plicatilisand C. cinereus, Psatyrella, Myceliophthora, e.g. M. thermonhila,Schytalidium, Phlebia, e.g., P. radita (WO 92/01046), or Coriolus, e.g.C.hirsutus (JP 2--238885).

The laccase or the laccase related enzyme may furthermore be one whichis producible by a method comprising cultivating a host cell transformedwith a recombinant DNA vector which carries a DNA sequence encoding saidlaccase as well as DNA sequences encoding functions permitting theexpression of the DNA sequence encoding the laccase, in a culture mediumunder conditions permitting the expression of the laccase enzyme, andrecovering the laccase from the culture.

Determination of Laccase Activity (LACU)

Laccase activity is determined from the oxidation of syringaldazin underaerobic conditions. The violet colour produced is photometered at 530nm. The analytical conditions are 19 μM syringaldazin, 23.2 mM acetatebuffer, pH 5.5, 30° C, 1 min. reaction time.

1 laccase unit (LACU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazin per minute at these conditions.

Enhancing Agents

The enhancing agent used in the present invention may be described bythe following formula: ##STR3## in which formula A is a group such as--D, --CH═CH--D, --CH═CH═CH═CH--D, --CH=N--D, --N═N--D, or --N═CH--D, inwhich D is selected from the group consisting of --CO--E, --SO₂ --E,--N--XY, and --N⁺ --XYZ, in which E may be --H, --OH, --R, or --OR, andX and Y and Z may be identical or different and selected from --H and--R; R being a C₁ -c₁₆ alkyl, preferably a C₁ -C₈ alkyl, which alkyl maybe saturated or unsaturated, branched or unbranched and optionallysubstituted with a carboxy, sulfo or amino group; and B and C may be thesame or different and selected from C_(m) H_(2m+1) ; 1≦m≦5.

In a preferred embodiment A in the above mentioned formula is --CO--E,in which E may be --H, --OH, --R, or --OR; R being a C₁ -C₁₆ alkyl,preferably a C₁ -C₈ alkyl, which alkyl may be saturated or unsaturated,branched or unbranched and optionally substituted with a carboxy, sulfoor amino group; and B and C may be the same or different and selectedfrom C_(m) H_(2m+1) ; 1≦m≦5.

In the above mentioned formula A may be placed meta to the hydroxy groupinstead of being placed in the para-position as shown.

In particular embodiments, the enhancing agent is acetosyringone,methylsyringate, ethylsyringate, propyl-syringate, butylsyringate,hexylsyringate, or octylsyringate.

The enhancing agent of the invention may be present in concentrations offrom 0.005-1000 μmole per g denim, referably 0.05-500 μmole per g denim,more preferably 0.5-100 μmole per g denim.

Stability of the Radical of the Enhancing Agent

Without being limited to any theory it is presently contemplated thatthere is a positive correlation between the half-life of the radicalwhich the enhancing agent forms in the relevant aqueous medium and itsefficiency in providing a bleached look in the colour density of thesurface of the dyed fabric together with the phenol-oxidizing enzymesystem, and that this half-life is significantly longer than thehalf-life of any of the substances selected from the group consisting ofp-hydroxycinnamic acid, 2,4-dichlorophenol, p-hydroxybenzene sulphonate,vanillin and p-hydroxybenzoic acid (i.e. the enhancing agents disclosedin WO 92/18683).

This invention therefore further relates to a process for providing ableached look in the colour density of the surface of dyed fabric, theprocess comprising contacting, in an aqueous medium, a dyed fabric witha phenol oxidizing enzyme system and an enhancing agent, wherein saidenhancing agent is capable of forming a radical having a half-life, insaid aqueous medium, which is at least 10 times longer than the radicalhalf-life of any one of the substances selected from the groupconsisting of p-hydroxycinnamic acid, 2,4-dichlorophenol,p-hydroxybenzene sulphonate, vanillin and p-hydroxybenzoic acid, testedin the same aqueous medium, in particular wherein said enhancing agentis capable of forming a radical having a half-life, in said aqueousmedium, which is at least 100 times longer than the radical half-life ofany one of the substances selected from the group consisting ofp-hydroxycinnamic acid, 2,4-dichlorophenol, p-hydroxybenzene sulphonate,vanillin and p-hydroxybenzoic acid, tested in the same aqueous medium.

As the half-life of the radical is dependent on, inter alia, the pH, thetemperature and the buffer of the aqueous medium, it is very importantthat all these factors are the same when the half-lifes of the radicalsof various enhancing agents are compared.

Industrial Applications

The process of the present invention is typically used in industrialmachines for making fabric look bleached. Normally, the process of theinvention will be performed on fabric already stonewashed, but theprocess may also be applied to fabric which has not undergone astonewashing process beforehand. Most commonly the fabric is added tothe machine according to the machine capacity per the manufacturer'sinstructions. The fabric may be added to the machine prior tointroducing water or the fabric may be added after water is introduced.The phenol oxidizing enzyme system and the enhancing agent of theinvention may be present in the water prior to adding the fabric or theymay be added after the fabric has been wetted. The phenol oxidizingenzyme system may be added simultaneously with the enhancing agent orthey may be added separately. After the fabric has been contacted withthe phenol oxidizing enzyme system and the enhancing agent of theinvention it should be agitated in the machine for a sufficient periodof time to ensure that the fabric is fully wetted and to ensure theaction of the enzyme system and the enhancing agent.

Absorbed Organic Halogens (AOX)

As a result of the chlorine-free bleaching process, AOX is expected tobe significantly lower when the process according to the invention isused compared to the conventional hypochlorite based process.

Strength Loss

The enzyme/enhancing agent bleaching process of the present inventionresults in a very specific attack on indigo and it is thereforecontemplated that the process does not result in a damage of the cotton,in particular no strength loss.

The invention is further illustrated in the following examples which arenot intended to be in any way limiting to the scope of the invention asclaimed.

EXAMPLE 1

The test procedure for denim bleaching was performed as described below:

Enhancing agents: Methylsyringate was obtained from Lancaster.Acetosyringone, p-hydroxybenzoic acid, p-hydroxy-benzene-sulfonate,2,4-dichlorophenol, vanillin and p-hydroxycinnamic acid were obtainedfrom Aldrich. Enzyme: Laccase derived from Trametes villosa (SP 504,available from Novo Nordisk A/S) was used.

Procedure: 18 ml 0.01 M B&R (Britt & Robinson) buffer (pH 4, 6, or 8)were added to a 50 ml conical flask. A magnet bar (4 cm) and a roundpiece of stone washed denim (3.5 cm diameter 0.4 g) were added to theflask together with 1 ml of the stock solution of the enhancing agent tobe tested and 1 ml of enzyme, giving a denim:liquor (w/w) ratio of 1:50;the final concentrations of the enhancing agent and the enzyme shown inTable 1-5 below.

The flask was incubated for 3 hours on a magnet stirrer in a water bath(50° C. and approximately 200 rpm). After the enzymatic bleaching, thedenim swatch was rinsed with distilled water and air dried, whereafterit was evaluated for the degree of bleaching. The evaluation wasperformed visually and by using a Minolta Chroma Meter CR200.

Evaluation: A Minolta Chroma Meter CR200 (available from Minolta Corp.)was used according to Manufacturer's instructions to evaluate the degreeof bleaching as well as to estimate any discoloration using the changein the colour space coordinates L*a*b* (CIELAB--system): L gives thechange in white/black at a scale of from 0 to 100, a gives the change ingreen (-a*)/red (+a*), and b gives the change in blue (-b*)/yellow (+b*). A decrease in L* means an increase in black colour (decrease of whitecolour) , an increase in L* means an increase in white colour (adecrease in black colour) , a decrease in a* means an increase in greencolour (decrease in red colour), an increase in a* means an increase inred colour (a decrease in green colour), a decrease in b* means anincrease in blue colour (a decrease in yellow colour), and an increasein b* means an increase in yellow colour (a decrease in blue colour).

The bleached stone washed denim swatches were compared to non--treatedstone washed swatches.

The Minolta Chroma Meter CR200 was operated in the L*a*b* coordinatesystem. The light source used was a CIE light standard C. Eachmeasurement was an average of 3 measurements. The instrument wascalibrated using a Minolta calibration plate (white). 10 non-treateddenim swatches were measured 2 times each and the average of thecoordinates L*a*b* were calculated and entered as a reference. Thecoordinates of the samples were then calculated as the difference(.increment.) of the average of 3 measurements on each swatch from thereference value of the coordinates L*a*b*.

                  TABLE 1                                                         ______________________________________                                        Table 1 shows Δ (L*/a*/b*) between a swatch                             treated with the tested system (different concentrations of                   laccase and 1000 μM acetosyringone˜50 μmole/g denim) and a        non-treated swatch at pH 4.                                                   0 μM      10 μM   100 μM                                             (0           (0.5       (5       1000 μM                                   μmole/g)  μmole/g)                                                                              μmole/g)                                                                            (50 μmole/g)                              ______________________________________                                        0                                  2.9/-0.5/0.4                               LACU/ml                                                                       0.1                                                                           LACU/ml                                                                       (78                                                                           μg/g)                                                                      1                                  5.8/-1.1/2.0                               LACU/ml                                                                       (780                                                                          μg/g)                                                                      5                                  6.3/-1.3/2.4                               LACU/ml                                                                       (3900                                                                         μg/g)                                                                      ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Table 2 shows Δ (L*/a*/b*) bewteen a swatch treated with                the tested system (different concentrations of laccase and                    acetosyringone) and a non-treated swatch at pH 6.                                        10 μM                                                                      (0.5       100 μM 1000 μM                                    0 μM    μmole/g)                                                                              (5 μmole/g)                                                                          (50 μmole/g)                               ______________________________________                                        0                                 2.9/-0.5/-0.3                               LACU/ml                           0.5/0.1/0.0                                 0.1            0.3/0.3/0.1                                                                              7.0/-1.0/1.7                                                                          11.7/-2.3/4.0                               LACU/ml                                                                       (78                                                                           μg/g)                                                                      1              0.5/0.2/0.2                                                                              7.8/-1.0/1.7                                                                          15.3/-2.7/5.5                               LACU/ml                           16.0/-2.7/5.9                               (780                                                                          μg/g)                                                                      5                                 19.2/-3.4/6.5                               LACU/ml                                                                       (3900                                                                         μg/g)                                                                      ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Table 3 shows Δ (L*/a*/b*) between a swatch treated with                the tested system (different concentrations of laccase and                    acetosyringone) and a non-treated swatch at pH 8.                                        10 μM                                                                      (0.5        100 μM  1000 μM                                  0 μM    μmole/g) (5 μmole/g)                                                                           (50 μmole/g)                             ______________________________________                                        0                                   1.7/0.0/0.5                               LACU/ml                                                                       0.1            0.1/0.3/-0.3                                                                              -0.5/0.4/-0.3                                                                          2.2/0.0/0.4                               LACU/ml                                                                       (78                                                                           μg/g)                                                                      1              -1.0/0.5/0.3                                                                              4.1/-0.6/2.2                                                                           4.1/-0.6/2.2                              LACU/ml                                                                       (780                                                                          μg/g)                                                                      LACU/ml                                                                       (3900                                                                         μg/g)                                                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Table 4 shows Δ (L*/a*/b*) between a swatch treated with                1000 μM methylsyringate (˜50 μmole/g) and laccase (1.0            LACU/ml                                                                       ˜780 μg/g) and a non-treated swatch at pH 4, 6 and 8.                        pH 4      pH 6       pH 8                                             ______________________________________                                        Methyl    8.2/-1.3/1.6                                                                              22.2/-3.2/6.6                                                                            4.5/-0.8/0.5                                 syringate:                                                                    (1000 μM˜                                                            50 μmole/g)                                                                Laccase:                                                                      (1.0 LACU/ml˜                                                           780 μg/g)                                                                  ______________________________________                                    

Visually a .increment.L around 5 gives a significant effect so it can beseen from the results presented in Table 1-4 that acetosyringone andmethylsyringate at pH 6 have a significant effect in bleaching denim.

                  TABLE 5                                                         ______________________________________                                        Table 5 shows Δ (L*/a*/b*) between a swatch treated with                the enhancing agents described in WO 92/18683 + laccase (0.1-                 1.0 LACU/ml corresponding to 78 μg enzyme protein/g denim-780              μg enzyme protein/g denim) and a non-treated swatch at pH 4, 6             and 8.                                                                        Tested System                                                                              pH 4        pH 6    pH 8                                         ______________________________________                                        p-Hydroxy-   0.85/       0.91/   -0.21/                                       benzoic acid:                                                                              -0.09/      -0.19/  0.24/                                        (1000 μM˜                                                                         0.61        -0.14   -0.17                                        50 μmole/g)                                                                Laccase:                                                                      (0.1 LACU/ml                                                                  ˜78 μg/g)                                                            p-Hydroxy-   -0.18/      0.33/   -0.51/                                       benzene-     0.14/       0.06/   0.17/                                        sulfonate:   -0.12       -0.22   -0.20                                        (1000 μM˜                                                            50 μmole/g)                                                                Laccase:                                                                      (0.1 LACU/ml                                                                  ˜78 μg/g)                                                            2,4-Dichloro-                                                                              0.64/       -0.19/  -0.54/                                       phenol:      -0.22/      -0.19/  0.16/                                        (1000 μM˜                                                                         0.5         0.57    -0.14                                        50 μmole/g)                                                                Laccase:                                                                      (0.1 LACU/ml                                                                  ˜78 μg/g)                                                            Vanillin:    -0.67/      0.28/   -0.38/                                       (1000 μM˜                                                                         -0.34/      -0.03/  -0.05/                                       50 μmole/g)                                                                             1.41        0.49    0.75                                         Laccase:                                                                      (1.0 LACU/ml                                                                  ˜780 μg/g)                                                           p-Hydroxy-   0.64/       4.47/   2.97/                                        cinnamic     -0.53/      -0.63/  -0.45/                                       acid:        1.62        3.88    0.79                                         (1000 μM˜                                                            50 μmole/g)                                                                Laccase:                                                                      (1.0 LACU/ml˜                                                           780 μg/g)                                                                  ______________________________________                                    

From the results presented in Table 5 it can be seen that none of theprior art described enhancing agents have any significant effect inbleaching the denim.

EXAMPLE 2 Comparison of performance in different buffers

Denim bleaching using methyl syringate (MS) was compared in thefollowing 3 buffers: Phosphate, oxalate, and acetate, all 0.01 M,prepared from Na₂ HPO₄ ×2H₂ O (pH adjusted with sulphuric acid), Na₂-oxalate (pH adjusted with sulphuric acid), and Na-acetatex3H₂ O (pHadjusted with sulphuric acid) respectively. Each buffer was prepared atpH 4.0, 5.0, 6.0, and 7.0 respectively.

300 ml of the buffer in question was added to a 1200 ml (total volume)stainless steel beaker together with 1 piece of stone washed denimweighing approximately 12 g (denim:liqour ratio=1:25); 1 ml of a 15 g/lMS (obtained from Lancaster) in 96% ethanol stock solution was added toeach beaker (corresponding to 236 μM or 5.9 μmole MS/g denim) togetherwith 0.132 ml of a 114 LACU/ml laccase stock solution (corresponding to0.05 LACU/ml or 19.5 μg enzyme protein/g denim). The laccase was derivedfrom Trametes villosa (TvL) and available from Novo Nordisk A/S (SP504).

The beakers were closed and processed at 60° C. for 30 minutes in aAtlas LP2 launder--ometer. Following processing, the denim swatches wererinsed in distilled water and air dried over--night, and the final pH ofthe bleaching liquor was measured.

When dry, the degree of bleaching of the denim was determined measuringthe absolute L*a*b* coordinates (average of 6 measurements) of thebleached denim as well as of the starting material from which.increment.(L*a*b*) was calculated. The results obtained are shown inTable 6 below.

                  TABLE 6                                                         ______________________________________                                        pH.sub.start                                                                            pH.sub.end                                                                           ΔL*    Δa*                                                                          Δb*                                  ______________________________________                                        0.01 M phosphate                                                              buffer                                                                        4.0       5.2    4.56         -0.71                                                                              1.24                                       5.0       5.3    6.11         -0.99                                                                              1.07                                       6.0       6.1    7.42         -1.37                                                                              0.74                                       7.0       7.0    0.16         0.04 0.20                                       0.01 M oxalate                                                                buffer                                                                        4.0       4.1    2.43         -0.28                                                                              0.44                                       5.0       5.3    6.40         -1.06                                                                              1.11                                       6.0       7.0    2.63         -0.38                                                                              0.81                                       7.0       7.7    1.44         -0.20                                                                              0.56                                       0.01 M acetate                                                                buffer                                                                        4.0       4.0    1.32         -0.27                                                                              0.46                                       5.0       5.0    4.96         -0.83                                                                              1.42                                       6.0       6.4    6.66         -1.26                                                                              0.90                                       7.0       7.4    0.89         0.00 0.39                                       ______________________________________                                    

From Table 6 it is seen, that there is no major influence on thebleaching process of the choice of buffer, besides the effect arisingfrom the drift in pH in the various buffers due to the poor buffercapacity of some of the buffers at some of the pH investigated. Furtherit is seen, that pH optimum lies in the range pH of 5.5-6.5, which is inaccordance with the results obtained in Example 1, Table 4.

EXAMPLE 3 Investigation of effect of varying concentration of methylsyringate (MS) and laccase

Denim bleaching using MS and 0.01 M phosphate buffer (prepared from Na₂HPO₄ ×2H₂ O, pH adjusted with sulphuric acid) was compared in the pHrange 5.0-6.5 for various dosages of MS and laccase.

300 ml of buffer was added to a 1200 ml (total volume) stain-less steelbeaker together with 1 piece of stone washed denim weighingapproximately 12 g (denim:liqour ratio=1:25), and 1 or 2 ml of a 15 g/lMS (obtained from Lancaster) in 96% ethanol stock solution was added toeach beaker (corresponding to 236 μM=5.9 μmole MS/g denim or 472 μM=11.8μmole MS/g denim) together with 0.132 or 0.264 ml of a 114 LACU/mllaccase stock solution (corresponding to 0.05 LACU/ml=19.5 μg enzymeprotein/g denim or 0.10 LACU/ml=39 μg enzyme protein/g denim). Thelaccase was derived from Trametes villosa (TvL) and available from NovoNordisk A/S (SP 504).

The beakers were closed and processed at 60° C. for 30 minutes in aAtlas LP2 launder-ometer. Following processing, the denim swatches wererinsed in distilled water and air dried overnight, and the final pH ofthe bleaching liquor was measured.

When dry, the degree of bleaching of the denim was determined measuringthe absolute L*a*b* coordinates (average of 6 measurements) of thebleached denim as well as of the starting material from which.increment.(L*a*b*) was calculated. The results obtained are shown inTable 7 below.

                  TABLE 7                                                         ______________________________________                                                  pH.sub.start                                                                         pH.sub.end                                                                           ΔL*                                                                              Δa*                                                                          Δb*                               ______________________________________                                        236 μM MS = 5.9                                                                        5.0      5.6    5.18   -0.88                                                                              1.10                                  μmole MS/g denim                                                                       5.5      5.8    5.44   -1.03                                                                              0.94                                  0.05 LACU/ml = 12.5                                                                       6.0      6.2    6.24   -1.13                                                                              0.78                                  μg enzyme protein/g                                                                    6.5      6.6    3.43   -0.67                                                                              0.52                                  denim                                                                         472 μM MS = 11.8                                                                       5.0      5.7    6.76   -1.20                                                                              1.34                                  μmole MS/g denim                                                                       5.5      5.9    6.93   -1.17                                                                              1.50                                  0.05 LACU/ml = 12.5                                                                       6.0      6.1    6.92   -1.28                                                                              0.97                                  μg enzyme protein/g                                                                    6.5      6.6    6.14   -1.07                                                                              0.69                                  denim                                                                         236 μM MS = 5.9                                                                        5.0      5.6    7.87   1.46 1.08                                  μmole MS/g denim                                                                       5.5      5.8    7.56   -1.45                                                                              0.90                                  0.1 LACU/ml = 25 μg                                                                    6.0      6.1    6.89   -1.35                                                                              0.75                                  enzyme protein/g                                                                          6.5      6.5    6.15   -1.11                                                                              0.46                                  denim                                                                         472 μM MS = 11.8                                                                       5.0      5.6    5.82   -0.96                                                                              1.13                                  μmole MS/g denim                                                                       5.5      5.8    7.32   -1.37                                                                              1.12                                  0.1 LACU/ml = 25 μg                                                                    6.0      6.1    7.04   -1.34                                                                              0.83                                  enzyme protein/g                                                                          6.5      6.6    6.24   -1.07                                                                              0.71                                  denim                                                                         ______________________________________                                    

From Table 7 it is seen, that increasing the concentration of either MSor laccase increases bleaching. Further, pH optimum is in the range5.5-6.0.

EXAMPLE 4 Denim bleaching using various enhancers

Enhancing agents: The enhancing agents were obtained from Lancaster(methylsyringate), Aldrich (acetosyringone), or were synthesized asdescribed in Chem. Ber. 67, 1934, p. 67.

Enzyme: Laccase derived from Trametes villosa (SP 504, available fromNovo Nordisk A/S) was used.

Procedure: 18 ml 0.01 M B&R (Britt & Robinson) buffer pH 4.0, pH 6.0, orpH 8.0 were added to a 50 ml conical flask. A magnet bar (4 cm), and around piece of stone washed denim (3.5 cm in diameter=0.4 g denim) wereadded to the flask together with 1 ml of the stock solution of theenhancing agent to be tested (0.02 M in 96% ethanol) and 1 ml of enzymestock solution (20 LACU/ml).

Summarizing the conditions used:

Denim:liquor ratio=1:50, 1.0 LACU/ml=780 μg enzyme protein/g denim, 1000μM.sup.˜ 50 μmole enhancing agent/g denim.

The flasks were incubated for 3 hours on a magnet stirrer in a waterbath (50° C. and approximately 200 rpm). After the enzymatic bleaching,the denim swatch was rinsed with water and dried in an oven atapproximately 110° C. for 15 minutes, whereafter it was evaluated forthe degree of bleaching. The evaluation was performed according to theprocedure mentioned in Example 1.

                  TABLE 8                                                         ______________________________________                                        Table 8 shows Δ (L*a*b*) between a swatch treated with the tested       system, and a non-treated swatch. Conditions: 0.01 M B&R buffer               pH 4.0, pH 6.0, or pH 8.0, denim:liquor ratio = 1:50, 1.0                     LACU/ml = 780 μg enzyme protein/g denim, 1000 μM˜50 μmole      enhancing agent/g denim. The flasks were incubated for 3 hours                on a magnet stirrer in a water bath (50° C. and approximately 200      rpm).                                                                         Enhancer pH 4.0       pH 6.0     pH 8.0                                       ______________________________________                                        Methyl-  3.9/-1.0/2.1 22.4/-4.3/5.7                                                                            2.0/-0.3/-0.0                                syringate                                                                     Ethyl-   7.6/-1.5/2.9 19.1/-3.6/5.8                                                                            1.2/0.1/-0.0                                 syringate                                                                     Propyl-  7.7/-1.7/3.2 20.9/-3.7/6.9                                                                            3.5/-0.4/1.2                                 syringate                                                                     Butyl-   11.1/-2.7/4.7                                                                              18.5/-3.6/7.1                                                                            1.3/-0.2/0.7                                 syringate                                                                     Hexyl-   9.3/-2.2/4.1 7.8/-1.9/3.1                                                                             0.3/0.1/0.5                                  syringate                                                                     Octyl-   8.2/-1.7/4.0 4.5/-1.4/2.9                                                                             1.8/-0.3/0.7                                 syringate                                                                     Aceto-   7.5/-1.8/4.5 17.9/-4.1/5.6                                                                            0.4/-0.2/1.1                                 syringone                                                                     ______________________________________                                    

We claim:
 1. A process for providing a bleached look in the colourdensity of the surface of dyed fabric, the process comprisingcontacting, in an aqueous medium, a dyed fabric with a phenol oxidizingenzyme system and an enhancing agent of the following formula: ##STR4##in which A is selected from the group consisting of --D, --CH═CH--D,--CH═CH═CH--CH--D, --CH═N--D, --N═N--D, and --N═CH--D, in which D isselected from the group consisting of --CO--E, --SO₂ --E, --N--XY, and--N+XYZ, in which E is selected from the group consisting of--H, --OH,--R, or --OR, and X, Y, and Z are selected from the group consisting of--H and --R in which R is a C₁ -C₁₆ alkyl, which is optionallysubstituted with a carboxy, sulfo or amino group; and B and C is C_(m)H_(2m+1), in which 1≦m≦5.
 2. The process according to claim 1, whereinX, Y, and Z are identical.
 3. The process according to claim 1, whereinX,Y, and X are different.
 4. The process according to claim 1, wherein Ris a KC₁ -C₈ alkyl.
 5. The process according to claim 1, wherein R is asaturated C₁ -C₁₆ alkly.
 6. The process according to claim 1, wherein Ris a nonsaturated C₂ -C₁₆ alkyl.
 7. The process according to claim 1,wherein R is a branched C₁ -C₁₆ alkyl.
 8. The process according to claim1, wherein R is an nonbranched C₁ -C₁₆ alkyl.
 9. The process accordingto claim 1, wherein B and C are the same.
 10. The process according toclaim 1, wherein B and C are different.
 11. The process according toclaim 1, wherein the fabric is dyed with a vat dye.
 12. The processaccording to claim 1, wherein the fabric is dyed with a vat dye selectedfrom the group consisting of indigo and thioindigo.
 13. The processaccording to claim 1, wherein the fabric is a cellulosic fabric or amixture of cellulosic fibres or a mixture of cellulosic fibres andsynthetic fibres.
 14. The process according to claim 1, wherein thefabric is denim.
 15. The process according to claim 14, wherein theconcentration of the phenol oxidizing enzyme corresponds to 0.001-10000μg of enzyme protein per g of denim.
 16. The process according to claim14, wherein the enhancing agent in the aqueous medium is present in aconcentration of from 0.005 to 1000 μmole per g denim.
 17. The processaccording to claim 1, wherein the fabric is denim dyed with indigo orthioindigo.
 18. The process according to claim 1, wherein the phenoloxidizing enzyme system is a peroxidase and a hydrogen peroxide source.19. The process according to claim 18, wherein the peroxidase ishorseradish peroxidase, soybean peroxidase or a peroxidase enzymederived from Coprinus, Bacillus, or Mvxococcus.
 20. The method accordingto claim 18, wherein the hydrogen peroxide source is hydrogen peroxideor a hydrogen peroxide precursor, a hydrogen peroxide generating enzymesystem, or a peroxycarboxylic acid or a salt thereof.
 21. The methodaccording to claim 18, wherein the hydrogen peroxide source isperborate, percarbonate, or an oxidase and its substrate.
 22. Theprocess according to claim 1, wherein the phenol oxidizing system is aperoxidase is derived from Coprinus cinereus, Coprinus macrorhizus,Bacillus pumilus, or Myxococcus virescens.
 23. The method according toclaims 1, wherein the aqueous medium contains H₂ O₂ or a precursor forH₂ O₂ in a concentration corresponding to 0.001-25 mM H₂ O₂.
 24. Theprocess according to claim 1, in which the phenol oxidizing enzymesystem is a laccase or a laccase related enzyme together with oxygen.25. The process according to claim 24, wherein the laccase is derivedfrom Trametes, Coprinus, or Myceliophthora.
 26. The process according toclaim 24, wherein the laccase is derived from Trametes villosa, Coprinuscinereus, or Myceliophthora thermophila.
 27. The process according toclaim 1, wherein the enhancing agent is selected from the groupconsisting of acetosyringone, methylsyringate, ethylsyringate,propylsyringate, butylsyringate, hexylsyringate, and octylsyringate.